Construction,Expression,Purification And Identification Of Recombinant Plasmids Based On Chicken Prolactin And Inhibin α Subunit Fragment

In construction of recombinant vaccines capable of inhibiting broodiness and increasing egg laying performance in broody prone avians, we choose the approach of producing recombinant proteins based on chicken prolactin and inhibin a subunit fragment A cDNA sequence coding mature chicken prolactin molecule was amplified by PCR and cloned into cloning sites of Bgl II and EcoR I in plasmid pRSET A, which generated a recombinant expressing plasmid pPRL-RSET. A fragment of chicken inhibin a subunit cDNA was also amplified and cloned into the sites of Nhe I and Xhol in plasmids pRSET A and pPRL-RSET. respectively to generate 2 recombinant expressing plasmids pINB-RSET and pINB-PRL. The accuracies of molecular clones in the recombinant plasmids were verified by PCR product lengths from specific primer combinations, by product length from specific restriction enzyme digestions and sequencing results of the plasmids. The plasmids were expressed in E.Coli BL21 (DE3) after induction by IPTG, and produced a proteinacous product showing the expected molecular size in SDS-PAGE. The two fusion proteins are inclusive. After induction with 0.01 mmol/L IPTG for 2h, pINB-PRL achieved the most expression; and after induction with 0.005mmol/L IPTG for 4h, pPRL-RSET expressed most The expressed products of pPRL-RSET and pINB-PRL were purified by Ni-NTA Aagrose column, and expressed the same migration as their corresponding products in expressed bacterial lysates both in SDS-PAGE and in Western-blot shown by anti-chicken prolactin antibody. These results demonstrated successful construction,exprssion,purification and identification of recombinant plasmids based on chicken prolactin and inhibin a subunit fragment.